The principal objective of this proposal is to contribute to an understanding of the catalytic activity of enzymes that act on proteins and of the factors that influence the efficiency of such enzymes. It is intended to use specially-designed oligopeptide substrates, to vary their structure systematically, and to attach fluorescent labels that can act as probes for conformational changes during the catalytic process. Kinetic measurements will be performed both by conventional and stopped-flow techniques, and studies will be conducted on the fluorescence polarization and lifetimes of the fluorescent labels in the enzyme-peptide complex. Also, the enzymes will be subjected to specific chemical modifications to examine their effects on both binding and catalytic efficiency. The enzymes that will be studied most intensively will be porcine gastric pepsin, beef spleen cathepsin D and Rhizopus- pepsin (both acid proteinases related to gastric pepsin), and papain. Because of the role of cathepsin D in protein breakdown in mammalian tissues, special attention will be devoted to the study of the control of the activity of this enzyme. It is also hoped to continue earlier work on chymotrypsin and on the partial proteolysis of lactic dehydrogenase in the context of our study of the relation of protein conformation to the catalytic efficiency of enzymes.